Stem cell factor (SCF) is a growth factor critical for normal hematopoiesis. The receptor for SCF is Kit, a receptor tyrosine kinase (RTK). Activating mutations in Kit are associated with mastocytosis, leukemia, germ cell tumors and gastrointestinal stromal cell tumors. Because of the importance of Kit in normal hematopoiesis, as well as its role in human disease, the objective of this project is to understand the mechanism of action of both normal and oncogenic forms of this receptor in hematopoietic progenitor cells and mast cells, two important target populations of SCF. One specific aim of this project is to define the biochemical mechanism of action of wild-type Kit. Specifically, we have focused on the role of the JAK/STAT pathway and Src family members in SCF-mediated responses. We have previously shown that Lyn is required for progenitor and mast cells to respond normally to SCF. This year we published work demonstrating that Lyn contributes to SCF-mediated responses through modulation of Jnks, PI3 kinase and Stat phosphorylation. Although the JAK/STAT pathway is critical in responses mediated by cytokine receptors, little is known about the role of this pathway in RTK-mediated responses. Using hematopoietic cells derived from JAK2-deficient embryos we have demonstrated that JAK2 contributes to SCF-mediated proliferation of primary progenitor cells. These are the first studies to demonstrate that a JAK family member contributes to responses mediated by a RTK. Lastly, SCF in combination with cytokines such as GM-CSF induces synergistic growth of hematopoietic progenitor cells. We have found that multiple mechanisms mediate synergy including interaction of Kit and the GM-CSF receptor, as well as synergistic activation of Erks. In addition our studies suggest that Kit makes both a structural as well as biochemical contribution to synergy with GM-CSF. A second specific aim of this project is to delineate signal transduction pathways mediating cellular transformation by D816V Kit, an oncogenic mutant found in patients with mastocytosis, leukemia and germ cell tumors. Previously we demonstrated the critical role of PI3 kinase in transformation of hematopoietic cells by D816V Kit. One signaling component often activated downstream of PI3K is PKC delta. Recently we found this PKC isozyme is activated after SCF treatment of wild type Kit and constitutively activated in cells expressing the D816V mutant. Interestingly, we demonstrated that this kinase contributes to negative regulation of wild type Kit compared to contributing to growth mediated by the Kit catalytic domain mutant. Thus, PKC delta may be a target in the treatment of disease associated with D816V Kit in humans. Lastly, high throughput screening studies were performed to identify compounds that directly inhibit the growth of the Kit catalytic domain mutant. These data could have important implications in designing treatment strategies of diseases associated with this mutation, such as mastocytosis and leukemia.